G protein coupled receptors (GPCRs) are a family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. GPCRs are found only in eukaryotes including yeast and animals. The ligands that bind and activate GPCRs include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, and are also the target of therapeutic drugs.
Vertebrate and C. elegans odorant receptors (ORs) are members of the G-protein coupled receptor (GPCR) family (Buck et al., 1991; Troemel et al., 1995). GPCRs are characterised by their seven transmembrane spanning domains with ligand binding domains inferred to be on the extracellular side of the membrane and G-protein binding domains on the intracellular side. When a receptor binds the ligand, a conformational change occurs in the receptor allowing it to activate a heterotrimeric G-protein (Kobilka et al., 2007). The activated G-protein can then activate signal transduction pathways such as the guanyl cyclase or phospholipase C pathways, transducing the signal to higher processing centres (Gaillard et al., 2004).
Forster resonance energy transfer, or simply resonance energy transfer (RET), is the non-radiative transfer of energy from an excited state donor molecule to a ground state acceptor molecule (Ghanouni et al., 2001). Energy transfer efficiency is dependent on the distance between the donor and acceptor, the extent of the spectral overlap and the relative orientation of the acceptor and donor dipoles. Previous cases where intramolecular RET has been used to monitor GPCR activation have employed a fluorescent donor and acceptor, a method referred to as fluorescence resonance energy transfer (FRET). In most cases both the fluorescent donor and acceptor are engineered variants of green fluorescent protein (GFP) from Aequoria victoria (Tsien, 1998). The most widely used FRET pair is cyan fluorescent protein (CFP) as the donor alongside yellow fluorescent protein (YFP) as the acceptor (Piston and Kremers, 2007) and this FRET system has previously been used to quantify direct ligand binding by a number of GPCRs (Lohse et al., 2003 and 2007; Vilardaga et al., 2003; Rochais et al., 2007; Lisenbee et al., 2007).
One method for monitoring receptor activation involves dual labelling a single GPCR with CFP and YFP at insertion sites within the third intracellular loop and C-terminus, respectively. Excitation of CFP with light at 436 nm causes CFP emission at 480 nm and FRET to YFP, which emits at 535 nm. The efficiency of FRET varies with the sixth power of the distance between donor and acceptor, providing an exquisitively sensitive indication of conformational changes in the GPCR. This was demonstrated with α2AR, parathyroid hormone receptor (PTHR), β1-AR and secretin receptors in intact cells. Interaction of the agonists noreadrenaline with α2AR (Lohse et al., 2003), parathyroid hormone with PTHR (Vilardaga et al., 2003), norepinephrine with β1-AR (Rochais et al., 2007) and secretin with secretin receptors (Lisenbee et al., 2007) changed the distance between CFP and YFP thus causing a change in FRET signal.
Replacement of the YFP acceptor with F1AsH, a fluorescein arsenical hairpin binder, in a FRET system (Hoffman et al., 2005; Nakanishi et al., 2006) resulted in a five-fold greater increase in agonist-induced FRET signal compared with the CFP/YFP system when used to monitor α2-adrenergic receptor activation (Nakanishi et al., 2006). However, F1AsH involves a more difficult labelling and washing procedure which has limited use in the wider research community. The CFP/YFP system remains the most frequently reported FRET system for monitoring intramolecular conformational change. A major disadvantage associated with FRET is the need for a light source to energise the donor fluorophore (Piston and Kremers, 2007). This causes unwanted direct excitation of the acceptor at the donor excitation wavelength (a problem referred to as ‘cross-talk”).
In bioluminescence resonance energy transfer (BRET), the donor fluorophore of FRET is replaced with a luciferase and the acceptor can be any suitable fluorophore. The use of a luciferase avoids the need for illumination as the addition of a substrate initiates bioluminescent emission and hence, RET. Two common implementations of BRET comprise Renilla luciferase (RLuc) with either coelenterazine h (BRET1; λem=˜475 nm) or coelenterazine 400a (Clz400a) substrate (BRET2; λem=˜395 nm) as the donor system coupled to either of the GFP mutants, YFP (BRET1; λem=˜530 nm) or GFP2 (BRET2; λem=˜510 nm). The BRET system offers superior spectral separation between the donor and acceptor emission peaks of ˜115 nm compared to ˜55 nm for the BRET1 system at the expense of the quantum yield (Pfleger and Eidne et al., 2006).
FRET with odorant receptors has only previously been demonstrated for Class A (a2-adrenergic and parathyroid hormone, (Vilardaga et al., 2003)) and Class B (secretin, (Lisenbee et al., 2007)) GPCRs. Unlike mammalian ORs, which belong to GPCR Class A, nematode ORs (Robertson, 1998 and 2001) belong to neither of these classes and are evolutionarily and structurally distinct. Furthermore, all ORs, including mammalian ORs, which sit within Class A of the GPCR superfamily, are atypical in respect of their expression. Generally these proteins cannot be functionally expressed other than in neurons derived from the chemosensory lineage. A number of accessory proteins have been identified whose presence is required for proper expression of mammalian and nematode ORs.
Thus, there is a need for suitably sensitive methods and molecules which enable the detection of compounds which bind G protein coupled receptors for use in, for example, biosensors.